Interference and RNA homologies of New Jersey serotype isolates of vesicular stomatitis virus and their defective particles

VIROLOGY

77, 490-500

(1977)

Interference and RNA Homologies of New Jersey Serotype Isolates of Vesicular Stomatitis Virus and Their Defective Particles W. M. SCHNITZLEIN Department

of Microbiology, Accepted

AND

University November

M. E. REICHMANN’ of Illinois,

Urbana,

Illinois

61801

lo,1976

Three New Jersey serotype isolates of vesicular stomatitis virus (VSV) were found to produce a population of defective interfering (DI) particles heterogeneous in size and in their RNAs. Only Ogden and Glasgow isolates produced DI particles containing a welldefined major RNA component. Annealing of Ogden mRNAs to Glasgow virion RNA revealed that the two isolates had approximately 24% homologies in their nucleotide sequences. One short DI particle, generated by the Glasgow isolate, contained only approximately 230 nucleotides homologous to the Ogden virion RNA. In spite of this lack of homology, the particle fully interfered with infections by Ogden virions. A previously described Indiana serotype DI particle (HR), which was shown to interfere heterotypitally with Prevec’s New Jersey virion, also contained only approximately 260 nucleotides homologous to the RNA of this isolate. None of the New Jersey DI particles exhibited the ability to fully interfere heterotypically with Indiana serotype virion infections. DI particles, generated by a given viral isolate, contained nucleotide sequences complementary to a part of their 30 S mRNAs, and the sequences of shorter particle RNAs were always contained in the RNA of the larger particles. In this respect, they resembled the nonheterotypically interfering DI particles of the Indiana serotype. INTRODUCTION

Recent investigations of a large number of defective interfering (DI) particles of vesicular stomatitis virus (VSV) of the Indiana serotype have made it possible to arrive at certain generalizations with respect to the nature of the conserved RNA sequences in these particles. Annealing experiments with various complementary viral mRNA species led to the conclusion that the RNA of all available particles, except one, originated at or near the 5’terminus of the viral genome and, depending on the size of the individual particle, extended towards the 3’-end of the 30 S mRNA cistron (Schnitzlein and Reichmann, 1976). The RNA of the one exceptional particle had very few (if any) nucleotide sequences in common with the other DI particle RNAs and contained all viral 13-18 S mRNA cistrons (Leamnson and Reichmann, 1974; Stamminger and ’ Address

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requests

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8 1977 by Academic

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nf rw-mvhwt,inn

in,qnv

to Dr.

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Lazzarini, 1974). This particle was also unique in its interfering abilities. While all the other DI particles interfere most efficiently with infections by their homologous Indiana serotype, the HR-DI particle interferes equally well heterotypically with infections of the distantly related New Jersey serotype (Huang and Wagner, 1966a; Prevec and Kang, 1970). It has also been demonstrated that the DI particle RNAs often contain self-complementary nucleotide sequences of varying lengths, occasionally resulting in hairpin-like structures (Lazzarini et al., 1975; Perrault, 1976). The complexity of the generation of these particles was further underlined by the report of the heterogeneous nature of DI particles found at early passages of some mutant and wild-type VSV (Indiana) infections and the preferential selection of one or two DI particles during later passages (Holland et al., 1976). The same isolates in our laboratory and in our

ISSN

0042-6622

RNA

HOMOLOGIES

BHK-21 cells (Glasgow), even when prepurified by repeated clonal isolations, reproducibly generated a single type of DI particle which contained one predominant species of RNA characteristic in size and nucleotide sequence (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976). We now find that several isolates of the New Jersey serotype generate two or more types of DI particles simultaneously, even though the virions were clonally prepurified six times. In one extreme case (Prevec’s isolate), the DI particle contained a heterogeneous population of RNAs which were so similar in size that a single predominant species could not be separated by polyacrylamide-gel electrophoresis. In spite of these differences, the New Jersey DI particles, which contained a major RNA component, were similar to the common Indiana DI particle RNAs insofar as their nucleotide sequences were also complementary to only the 30 S mRNA species and the sequences of the shorter RNAs were contained in the longer RNAs. Also, these particles were unable to interfere heterotypically with infections by virions of the Indiana serotype. Various wild-type isolates of the Indiana serotype, previously investigated in our laboratory, did not show a lack of homology in their nucleotide sequences by annealing techniques (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976). We now find that two isolates, Ogden and Glasgow, both classified as New Jersey serotypes, exhibit only approximately 25% homologies in their RNA sequences. A third isolate (Prevec’s) was indistinguishable from Ogden. A short DI particle, isolated from BHK-21 cells infected with,the Glasgow New Jersey virus, interfered equally efficiently with infections by either Ogden or Glasgow virions. This interference occurred even though the DI particle RNA and the Ogden virion RNA had a homology equivalent to no more than 240 nucleotides. A maximum homology of this magnitude was also found between the HR-DI particle (Indiana) RNA and Prevec’s (New Jersey) virion RNA. In this case, the HR-DI particle (Indiana) also interfered as efficiently with

OF

NEW

JERSEY

491

VSV

the heterologous New Jersey virion as with its own homologous Indiana virion (Prevec and Kang, 1970). MATERIALS

AND

METHODS

Cells. Monolayers of BHK-21 Cl3 cells (Macpherson and Stoker, 1962) and mouse L cells (a gift from Dr. Holland) were propagated as described previously (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976). Preparation and purification of viruses and DI particles. Three wild-type isolates of the New Jersey serotype of VSV were kindly provided by Drs. Holland (Ogden, 0) (Perrault and Holland, 1972), Prevec (P) (Prevec and Kang, 1970), and Pringle (strain M, Glasgow, G) (Pringle et al., 1971). A temperature-sensitive (ts) mutant Cl, originally derived from the Glasgow wild type, was also obtained from Dr. Pringle (Pringle et al., 1971). The three isolates of the Indiana serotype (Gla, MS, and HR) were described previously (Schnitzlein and Reichmann, 1976). Viral inocula, free of DI particles, were prepared by sixfold clonal isolation followed by a single enrichment step (Stampfer et al., 1971). The original inocula were passaged undiluted in order to produce high yields of DI particles. Virus and DI particles were grown in BHK-21 cells and purified as described (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976) except that 3E buffers were used. Znterference assays. Prior to infection of cells, 20 ~1 of 0.1 M MgCI, -6H,O was added to DI particle preparations in order to remove the EDTA. Cell monolayers in square bottles (3 x lo6 cells) were coinfected (or mock infected) with DI particle preparations and with infectious virions (2-5 PFU/cell) for 30 min. After absorption, the cell monolayers were washed twice with 5 ml of sterile saline (0.85% NaCl) and covered with 5 ml of either BHK-21 medium (Leamnson and Reichmann, 1974) without tryptose phosphate (BHK-21 cells) or minimal essential medium (Schnitzlein and Reichmann, 1976) (L cells). After 9 hr at 39”, samples from duplicate cell cultures were removed and

492

SCHNITZLEIN AND REICHMANN

stored at -70” for subsequent plaque assays. Plaque assays were performed in duplicate as described previously (Schnitzlein and Reichmann, 1976).

RESULTS Properties of New Jersey DI Particles Their RNAs

and

Three isolates were used in these studies, G, 0, and P (see Materials and Methods). The G and 0 virions generated short Jersey virus-induced cytoplasmic RNA and long DI particles which were purified was prepared as previously described separately and designated GS and OS and (Schnitzlein and Reichmann, 1976). The GL and OL, respectively. None of these profiles on gels and gradients were similar New Jersey DI particles had the ability to to those of the Indiana serotype cytoplasinterfere with Indiana serotype (Gla) virus mic RNA (Schnitzlein and Reichmann, to the same extent as with New Jersey (PI 1976). Due to the low incorporation of serotype virus. This is shown in Table 1, r3H]uridine into P and 0 viral mRNAs, where the reduction in infectious progeny these were 3H labeled in both cytidine and is expressed as a function of concentration uridine. G viral 30 S mRNA was made of DI particles in the inoculum. A similar from cells infected with wild-type virus, observation with G-D1 particles has been while 13-18 S mRNAs were isolated from reported (Crick and Brown, 1973). As in cells infected with the ts mutant Cl (Printhe case of Indiana DI particles (except gle et al., 1971). This mutant has been HR), heterotypic interference never exshown to produce approximately eightfold ceeded 10% of the value for homotypic inmore viral 13-18 S mRNAs than G virus, terference. However, unlike the case of the when the infected cells were incubated at Indiana particles (except HR) (Prevec and nonpermissive temperature (Lesnaw and Kang, 1970; Schnitzlein and Reichmann, Reichmann, 1975). 1976), this heterotypic interfering ability Particle-bound RNA was isolated by was also expressed in L cells (Table 2). phenol extraction in 3E buffer (Kirby, The RNAs, extracted from purified DI 1965). Indiana serotype DI particle RNAs particle preparations, were examined by ([14Cluridine labeled) were further puripolyacrylamide-gel electrophoresis (Fig. fied by centrifugation in sucrose gradients, 1). The P-D1 particle RNA was very heterwhile New Jersey serotype DI particle ogeneous and electrophoresed as a broad RNAs were purified by elution from polyband near the position of the 28 S riboacrylamide gels as described previously somal RNA marker (Fig. la, 8-24 mm). (Schnitzlein and Reichmann, 1976). Prior The OS-D1 particles, on the other hand, to gel elutions, specific activities of 14C- contained two separable major RNA spelabeled RNAs were determined from spec- cies (Fig. lb), one at 23-27 mm (OS,) and trophotometric and radioactive measurethe other at 30-33 mm (O&j. Likewise, ments. An extinction coefficient of 25 for a two major RNA species were isolated from 1-mg/ml RNA solution at 260 nm and a OL-DI particles (Fig. Id), one at 13-14 mm light path of 1 cm was used in these cal(OL,) and the other at 16-17 mm (OL,). culations. After elution and ethanol pre- These two RNA species were separated by cipitation, RNA concentrations were based prolonged electrophoresis (Fig. Id, inset at on the number of counts eluted and the upper right-hand corner). The GL-DI parpredetermined specific activity. ticle RNA was composed of two major comGel electrophoresis and annealing of ponents (Fig. 14, one at 11-15 mm (GL,) RNA species. Electrophoresis in 1 x &cm, and the other at 19-23 mm (GL,). The GS2.4% polyacrylamide gels was performed DI particle contained a single RNA species at 12 mA/gel for 3.5 or 6 hr as described (Fig. lc, 33-36 mm). previously (Unger and Reichmann, 1973). The RNA species, isolated from these DI The annealing experiments were carried particle preparations, were probably conout as described by Leamnson and Reichtained in various DI particles which were mann (1974). not separated by rate zonal centrifugation

Preparation and purification of cytoplasmic and particle-bound RNAs. New

RNA HOMOLOGIES

because of their similarity in size. Previous experience with DI particles has shown that their RNA content is reflected in the size of the particles (Huang and Wagner, 1966b; Nakai and Howatson, 1968; Petric and Prevec, 1970; Reichmann et al., 1971). In view of this, it is not likely that multiple nucleocapsid species would be contained in the same particle without seriously affecting its sedimentation coefficient. Annealing of Particle-Bound RNA with Viral-Induced Cytoplasmic RNA

The nucleotide sequences of the New Jersey DI particle RNAs were characterized by annealing with various mRNA species. Since 0 and G virion RNAs did not contain completely homologous nucleotide sequences (see Table 31, annealings between the major RNA species of 0- and GDI particles and their respective viral mRNAs were performed. P-D1 particle RNA was not used in these studies due to its very heterogeneous nature. Because of the high self-annealing (6080%) of the O-D1 particle RNAs, a constant amount of 3H-labeled 0 viral mRNAs was annealed with excess particle RNAs. Very little (if any) of the viral 13-18 S mRNAs were made ribonuclease resistant by annealing with any of the O-D1 particle RNAs (Fig. 2a, open triangles and solid circles). However, each of the O-D1 particle RNAs were complementary to a portion of the viral 30 S mRNA (Figs. 2b and c; open triangles, circles, and squares and solid circles). As expected, virion RNA annealed to approximately 90% of both mRNA preparations (Figs. 2a and b, solid squares). When the viral 30 S mRNA was annealed with a mixture of both OSz- and OL,-DI particle RNAs (Fig. 2c, solid triangles), the results were similar to those obtained by using only OL,-DI particle RNA (Fig. 2c, open triangles). This suggested that the nucleotide sequences of the shorter DI particle RNA (OS,) were contained in those of the longer RNA (OL,). The nucleotide sequences of G-D1 particle RNAs were similarly examined by annealing to 3H-labeled G viral mRNA. In contrast to viral RNA which was comple-

OF NEW JERSEY

493

VSV TABLE

1

HOMOTYPIC AND HETEROTYPIC INTERFERING ABILITY OF DI PARTICLES OF THE NEW JERSEY SEROTYPE OF VSV IN BHK-21 CELLS”

DI particles

Dilution of DI particles

Log reduction of infectivity (PFUlmllb

New Jer- Indiana sey sero- serotype type GS Undiluted 1.56 0.29 1:2 1.30 0.00 I:5 0.99 0.03 0.80 0.00 1:lO GL Undiluted 1.77 0.40 I:2 1.52 0.36 1:5 1.04 0.24 0.83 1:lO 0.12 OS Undiluted 1.75 0.30 1:2 1.44 0.21 15 1.09 0.15 1:lO 0.68 0.00 OL Undiluted’ 3.52 1.75 1:2 2.80 1.18 15 1.67 0.45 1:lO 1.44 0.16 P Undiluted 1.44 0.13 1:2 0.75 0.12 1:5 0.59 0.03 0.00 1:lO 0.42 n Samples were withdrawn 9 hr after infection and assayed for plaque-forming ability (see Materials and Methods). b Results are expressed as the log difference of infectious particles produced from BHK-21 cells coinfected with DI and virion particles as compared to cells infected with virion only. In the absence of DI particles, the yields of both serotypes were approximately 2 x lo8 PFU/ml. C Very much higher yields of this DI particle resulted in considerably greater concentrations of the undiluted preparation as compared to all other DI particles presented here.

mentary to more than 90% of the viral 1318 S mRNAs (Fig. 3a, solid squares), the G-D1 particle RNAs were complementary to very few (if any) of the nucleotide sequences of these mRNAs (Fig. 3a, open squares, circles, and triangles). All three of the DI particle RNAs rendered part of the viral 30 S mRNA ribonuclease resistant (Fig. 3b, open circles, squares, and triangles). The preparation of 30 S mRNA, used in the experiments of Fig. 3b, showed approximately 18% self-annealing. This was probably due to contamination with

494

SCHNITZLEIN

AND

labeled viral RNA and is reflected in the incomplete annealing (84%) to excess unlabeled viral RNA (Fig. 3b, solid squares). As in the case of the O-D1 particles, mixed annealing data with GS- and GL,-DI particle RNAs (Fig. 3b, solid triangles) cointided with the GL, annealing data (Fig. 3b, open triangles), indicating that the RNA of the longer particle (GL,) contained TABLE HOMOTYPIC

Dilution

2

AND HETEROTYPIC OF OL-DI PARTICLES

of OL-DI particle

Log

INTERFERING IN L CELLSO

reduction of infectivity (PFU/mDb

New Jersey serotype Undiluted 1:2 1:5

4.11 3.71 1.50 0.90

1:lO I’ The experimental described in footnote a ’ Results are shown tious particles produced DI and virion particles with virion only. In the yields of both serotypes PFU/ml

ABILITY

Indiana serotype 2.79 2.39 0.40 0.33

conditions were the same as to Table 1. as the log difference of infecfrom L cells coinfected with as compared to cells infected absence of DI particles, the were approximately 4 x 10’

(a)

REICHMANN

all the nucleotide sequences of the shorter particle RNA (GS1. Annealing of a constant amount of 3Hlabeled particle-bound RNA with increasing amounts of viral mRNA was also performed with the G-D1 particle RNAs. The mRNA preparations were examined for the presence of all species by annealing with virion RNA. The results are shown in Fig. 4a (open triangles) and demonstrate that no nucleotide sequences appeared to have been absent, since over 90% of the viral RNA was rendered ribonuclease resistant. The 13-18 S mRNAs annealed to approximately 50% of the viral RNA (Fig. 4a, open circles), analogous to results obtained with the Indiana serotype (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976). However, the annealing curve obtained with excess 30 S mRNA (Fig. 4a, solid circles) did not plateau, probably because of low-level contamination with 13-18 S mRNAs. At least 85% of the nucleotide sequences contained in the G-D1 particle RNAs were complementary to the 30 S mRNA species (Figs. 4b, c, and d, solid circles). The slight increase of ribonuclease resistance of the G-D1 particle RNAs in the presence of excess 13-18 S 1

4 (b)

3285

285

Dvhnce

Migrated

ISS

(mm)

FIG. 1. Polyacrylamide-gel electrophoresis of DI particle RNA species. The RNA was extracted from purified 3H-labeled DI particles as described in Materials and Methods. Ribosomal RNA was isolated from W-labeled BHK-21 cells and coelectrophoresed with the DI particle RNAs on 2.j % polyacrylamide gels at 12 mA/gel for 3.5 hr as described previously (Unger and Reichmann, 1973). The following isolates are shown: (a), P; (b), OS; (c), GL (left ha10 and GS (right half); (d), OL. The inset in the upper right-hand corner of (d) is a gel profile of OL-DI particle RNA after electrophoresis for 6 hr.

RNA TABLE ANNEALING

Amount w

G Virus

GS-DI

GL,-DI

BHK-21 RNA

mRNA

ribosomal

used

(cpm)

NEW

JERSEY

495

VSV

WITH RNAs

VARIOUS

Radioactivity in 0 mRNA after RNAse treatment” (cpm)

RNA

0 Virus

OF

3

OF 0 VIRAL mRNAs VIRION AND DI PARTICLE

‘be

HOMOLOGIES

0.05 0.10 0.20 0.50 0.05 0.10 0.20 0.50 0.05 0.10 0.20 0.50 0.05 0.10 0.20 0.50 0.05 0.10 0.20 0.50

13-18

S

867 1057 1041 1088 210 363 303 296 0 0 0 5 0 0 0 0 1129

30 s 651 781 860 834 33 120 112 141 6 32 22 32 40 43 67 65 0 0 0 0 910

’ DI particle RNAs were purifed by elution from polyacrylamide gels and were weakly labeled with lL4Cluridine (approximately 700 cpmlyg) for identification purposes (see Materials and Methods). The experimental procedure was the same as described in the legend to Fig. 2. Results are expressed as “H counts per minute in 0 viral mRNA species resistant to ribonuclease after annealing with the indicated amounts of RNA. The self-annealing of the mRNA species (14 cpm, 13-18 S; 48 cpm, 30 S) was subtracted from all values. 3H radioactive counts were corrected for a spillover from the low level of ‘*C counts.

mRNAs (Fig. 4b, c, and d, open circles) was due either to a short complementary sequence to these mRNAs or to contamination. In any case, the G-D1 particle RNAs were mainly composed of a part of the viral 30 S mRNA cistron. Interference between DI Particles and Virion Isolates of Limited RNA Homologies Isolates of the Indiana serotype, obtained from various laboratories, were

5kji

0

01

02

0.3

04

05

RNA (pg)

FIG. 2. Annealing of 0 viral mRNA species to variable amounts of 0 virion and DI particle RNA species. DI particle RNAs’ were purified by elution from polyacrylamide gels and were weakly labeled with [‘*Cluridine (approximately 600 cpm/pg) for identification purposes (see Materials and Methods). Annealing followed by ribonuclease digestion and precipitation with trichloroacetic acid was done as previously described (Leamnson and Reichmann, 1974). 0 viral 13-18 S (a) or 30 S (b and c) mRNA species were annealed to 0 virion RNA (-U-m-), OS, C-O-O-), OS2 (-4-O-), OL, (-A-A-,, OL, (-O-O-), or a 1:l mixture of OS, and OL, (-A-A-) DI particle RNAs. The baseline c-----l was obtained by annealing with BHK-21 ribosomal RNA. Annealing of the 13-18 S mRNA species to OS,- and OL,-DI particle RNA produced results identical to the base line and were omitted for the sake of clarity. 3H radioactive counts were corrected for a spillover from the low level of 14C counts.

found to be homologous in the nucleotide sequences of their RNA (Leamnson and Reichmann, 1974; Schnitzlein and Reichmann, 1976). In contrast, G New Jersey viral RNA exhibited only partial homologies with the 0 New Jersey isolate, when examined by annealing with 0 mRNAs. This is shown in the data in Table 3. When excess G viral RNA was annealed with 3Hlabeled 0 viral mRNAs, approximately

SCHNITZLEIN

496

--------____01.1

01

02

03

RNA (pg)

AND REICHMANN

25

04

05

FIG. 3. Annealing of G viral mRNA species to variable amounts of G virion and DI particle RNA species. DI particle RNAs were labeled, purified, and annealed as described in Fig. 2 and in Materials and Methods. G viral 13-18 S (a) or 30 S (b) mRNA species were annealed to G virion RNA (44-1, GS (-O-O-), GL, (-A-A-), GLZ (-O-O-), or a 1:l mixture of GS and GL, (-A-A-) DI particle RNAs. The baseline (-----I was obtained by annealing with BHK-21 ribosomal RNA. 3H radioactive counts were corrected for a spillover from the low level of j4C counts.

32% of the 13-18 S mRNAs and 16% of the 30 S mRNA became ribonuclease resistant. The homologous 0 viral RNA was complementary to over 90% of these mRNAs. G-D1 particle RNAs were also annealed with 0 viral mRNA in order to determine the extent of their homologies (Table 3). GL,- and GS-DI particle RNAs annealed to approximately 7% and 3.5%, respectively, of the 30 S mRNA. The GSDI particle RNA showed no complementarity to the 13-18 S mRNA species. Assuming the molecular weight of New Jersey viral RNA to be 4.5 x lo6 @chaffer and Soergel, 1972), the 30 S mRNA would correspond to approximately 6650 nucleotides. Based on these calculations, the GS-DI particle RNA and 0 viral RNA would have

common sequences of approximately 230 nucleotides. In spite of the limited homology, GS-DI particles (at all concentrations) interfered with infections of 0 New Jersey virus to the same extent as with those of homologous G New Jersey virus (Table 4). It should be noted that the same GS-DI particle preparation had very little effect on infections with the Indiana serotype (see Table 1). Since the HR-DI particle (Indiana) had the ability to interfere with virus of the heterologous New Jersey serotype (Prevec and Kang, 1970; Schnitzlein and Reichmann, 19761, the extent of homology between the RNAs of this DI particle and the New Jersey virus was determined by annealing to P viral mRNAs. These data are shown in Table 5. The HR-DI particle RNA was complementary to approximately 3.9% of the 13-18 S mRNA species. This complementarity is equivalent to approximately 260 nucleotides, which is very similar to the extent of homology between GS-DI particle and 0 viral RNA. These data suggest that a relatively short nucleotide sequence may be required for complete interference. Table 5 also shows that the MS-D1 particle of the Indiana serotype, which does not interfere with the P New Jersey isolate, has little complementarity (0.8%) to the 30 S mRNA. The Gla (Indiana) viral RNA annealed to 3.1 and 1.7% of the 13-18 S and 30 S P viral mRNA species, respectively. A similar degree of homology between Indiana and New Jersey serotypes of VSV was also reported by Repik et al., (1974). A higher complementarity observed by Prevec (1974) was probably due to incomplete digestion of the annealed products, since only ribonuclease A and not a mixture of ribonuclease A and T, was used in the latter experiments. DISCUSSION

The reproducible selection of a single type of DI particle, observed in infections with the Indiana serotype of VSV (Huang and Wagner, 1966b; Nakai and Howatson, 1968; Petric and Prevec, 1970; Reichmann et al., 1971), was recently questioned by the detection of two or more DI particles in

RNA

HOMOLOGIES

OF

NEW

JERSEY

497

VSV

(b)

c +

5

25

k s

O

t

,

0

IO

20

20

40 RNA

30

40

50

60

so

100

lpll

0

20

60 40 RNA (pl)

SO

100

FIG. 4. Annealing of G virion and DI particle RNA species to variable amounts of G viral mRNA species. The experimental procedure was the same as described in the legend to Fig. 2 and in Materials and Methods. 3H-labeled DI particle RNAs were purified by elution from polyacrylamide gels. G virion RNA (a), GS (b), GL, (c) or GL, (d) DI particle RNA was annealed to either G viral 13-18 S (-0-O-l or 30 S (-0-O-l mRNA species and to 0.02 pg/pl of BHK-21 ribosomal RNA (-0-O-X G virion RNA (a) was also annealed to a combination of both G virion 13-18 S and 30 S mRNA species (-A-A-). 3H radioactive counts were corrected for a spillover from the low level of 14C counts (lo-15 cpm/Fl, prior to annealing) in the mRNA species.

TABLE

4

INTERFERING ABILITY OF GS-DI PARTICLES WITH Two ISOLATES OF THE NEW JERSEY SEROTYPE IN BHK-21 CELLSa

Dilution of DI particle

Undiluted 1:2 I:5 1:lO

Log

reduction of infectivity (PFUlmDb

Glasgow

Ogden

1.53 1.02 0.71

1.49 1.12 0.89

0.47

0.52

o The experimental conditions were the same as described in footnote a to Table 1. b Results are shown as the log difference of infectious particles produced from BHK-21 cells coinfected with virion and DI particles as compared to cells infected with virion particles only. In the absence of DI particles, the yields of both types of New Jersey serotype virions were approximately 3 x 10’ PFU/ml.

infections of various clonally purified Indiana isolates (Lazzarini et al ., 1975; Holland et al., 1976). Heterogeneity of New Jersey DI particle RNAs has been reported

(Perrault and Holland, 1972). However, this preparation was not obtained by repeated clonal isolation. The data, presented in this paper, show heterogeneity to be a common feature of New Jersey isolates, even after a sixfold clonal purification. In the isolate obtained from Dr. Prevec’s laboratory, the generation of several, similar-sized DI particle RNA species prevented an effective separation either by rate zonal centrifugation of the particles themselves or by polyacrylamide-gel electrophoresis of their RNAs. Therefore, a study of these particles had to be abandoned. Since the mechanism of the generation of DI particles is poorly understood, it is not clear what caused the greater heterogeneity of some isolates as compared to others. Previously, we reported the absence of any correlation between the complementation group of some Indiana ts mutants and the type of DI particle which they generated (Reichmann et al., 1971). This lack of correlation suggested the absence of a genetic basis in the selection

498

SCHNITZLEIN TABLE

ANNEALING

P (N.J.)

Virus

G (Ind.)

Virus

MS (Ind.1

DI

HR

DI

BHK-21 RNA

mRNA

5

OF P mRNAs WITH VARIOUS VIRION AND DI PARTICLE RNAs Radioactivitv in P mRNA after RNA RNAse treatment? Amount b%)

‘Me

(Ind.)

AND

ribosomal

used

(cpm)

0.25 0.50 1.00 1.75 2.50 0.25 0.50 1.00 1.75 2.50 0.25 0.50 1.00 2.50 0.25 0.50 1.00 2.50 0.25 0.50 1.00 2.50

13-18 6150 8888 9733 10177 10203 170 226 329 275 290 13 13 14 0 130 364 314 423 0 0 0 0 10775

S

30 s 6718 7598 7576 8348 8561 35 145 113 99 158 52 36 61 81 91 68 62 64 32 39 7 44 9232

” DI particle RNAs were purified by centrifugation in sucrose gradients and were weakly labeled with [L4C1uridine (approximately 875 cpm/pg) for identification (see Materials and Methods). The experimental procedure was the same as described in the legend to Fig. 2. Results are expressed as 3H counts per minute in P viral mRNA species resistant to ribonuclease after annealing with the indicated amounts of RNA. The self-annealing of the ‘mRNA species (418 cpm, 13-18 S; 150 cpm, 30 S) was subtracted from all values. 3H radioactive counts were corrected for a spillover from the low level of “C counts.

process of DI particles. However, the possibility of silent mutations in the 30 S mRNA cistron, which might influence this phenomenon, cannot be altogether excluded. Members of the VSV group have been classified on the basis of antigenic relatedness (Federer et al., 1967). Since antigenicity is determined by the G and N proteins

REICHMANN

(Cartwright and Brown, 1972), this type of classification would predict RNA homologies affecting only these two cistrons, or approximately 3470 nucleotides (Rose and Knipe, 1975). This constitutes approximately 30% of the 3.8 x lo6 daltons of the viral genome (Repik and Bishop, 19731. It is therefore not surprising that variations in other cistrons would go undetected by the antigenic classification. The large lack of homology (90%) between the Indiana and Coca1 subgroups of the Indiana serotype (Repik et al., 1974) has demonstrated the lack of correlation between these two types of classification. The two isolates of the New Jersey serotype, 0 and G, which showed limited homologies in their nucleotide sequences, may also show antigenic differences by more sensitive techniques. A subdivision of the New Jersey serotype into subgroups, like the Indiana serotype, may be useful. In any case, it is important in future work that the source of the viral isolate be specified. Five types of DI particles, generated by the three New Jersey isolates, were found to be unable to interfere heterotypically with infections of the Indiana serotype. The high specificity of interference, observed for DI particles of Indiana serotype, seemed to be characteristic of the New Jersey DI particles also. Moreover, the nature of the conserved sequences in the DI particle RNAs of the two serotypes also appeared to be similar. Both groups contained sequences which were complementary to a part of their respective 30 S mRNAs. In addition, the longer DI particle RNAs contained all the nucleotide sequences of the shorter RNAs. These findings may reflect the necessity to conserve a certain nucleotide sequence, near or at the Y-terminus of the viral genome, which is required for interference. The shortest DI particle RNA of the Indiana serotype, ts G 31, established the upper limit of this sequence at approximately 1000 nucleotides (Leamnson and Reichmann, 1974). The present data on the extent of homology between GS-DI particle and 0 viral RNA limited this sequence to approximately 230 intracistronic nucleotides. A similar degree of homology was also found between the

RNA

HOMOLOGIES

RNAs of the heterotypically interfering HR-DI (Indiana) particle and the P (New Jersey) virion. However, these homologies were not present in the same cistrons. All the homologies of the HR-DI particle RNA with P viral RNA corresponded to the 1318 S mRNA cistrons. Its homologies with the viral 30 S mRNA cistron did not exceed those of the noninterfering MS (Indiana)DI particle (Table 5). Conversely, the GSDI particle RNA homologies with 0 viral RNA were all contained in the 30 S mRNA cistron, and none was found in the 13-18 S mRNA cistrons. These data might suggest that the mechanism of interference of the HR-DI particle is different from that of other DI particles. The phenomenon of heterotypic interference and the generation of DI particles from the 13-18 S mRNA cistrons of viral RNA, which is found in the HR-DI particle, seem to be rare events. So far, no corresponding New Jersey DI particles or other Indiana DI particles have been found. Alternately, it is possible that none of these sequences is important in interference. It should be noted that the data presented here cannot distinguish between one contiguous homologous nucleotide sequence and several scattered ones. Also, the experimental error in the determinations of RNA homologies between the GSDI particle and 0 virion is rather large. It is possible that the real site of interference involves extracistronic regions which would escape detection by annealing with mRNA species. Since over 90% of virion RNA anneals to viral mRNAs, these extracistronic regions would probably not be very long. Preliminary analyses by fingerprinting have suggested the presence of such extracistronic sequences. This problem is currently being elucidated. ACKNOWLEDGMENTS We thank Drs. Holland, Pringle, and Prevec for their VSV isolates. Ms. P. Bay and Ms. L. Erickson’s excellent technical assistance was appreciated. This work was supported in part by Grants USPH AI 12070 and NSF GB 34171. One of the authors (W. S.) was a recipient of a Public Health Service Predoctorate Traineeship from a Microbiology Training Grant (GM 00510).

OF

NEW

JERSEY

VSV

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REFERENCES B., and BROWN, F. (1972). Serological relationship between different strains of vesicular stomatitis virus. J. Gen. Virol. 16, 391-398. CRICK, J., and BROWN, F. (1973). Interference as a measurement of cross-relationship in the vesicular stomatitis group of rhabdoviruses. J. Gen. Viral. 18, 79-82. FEDERER, K. B., BURROWS, R., and BROOKSBY, J. B. (1967). Vesicular stomatitis virus-The relationship between some strains of the Indiana serotype. Res. Vet. Sci. 8, 103-117. HOLLAND, J. J., VILLARREAL, L. P., and BREINDL, M. (1976). Factors involved in the generation and replication of rhabdovirus defective T particles. J. Virol. 17, 8054315. HUANG, A. S., and WAGNER, R. R. (1966al. Defective T particles of vesicular stomatitis virus. II. Biologic role in homologous interference. Virology 30, 173-181. HUANG, A. S., and WAGNER, R. R. (1966b). Comparative sedimentation coefficients of RNA extracted from plaque-forming and defective particles of vesicular stomatitis virus. J. Mol. Biol. 22, 381-384. KIRBY, K. S. (1965). Isolation and characterization of ribosomal ribonucleic acid. Biochem. J. 96, 266269. LAZZARINI, R. A., WEBER, G. H., JOHNSON, L. D., and STAMMINGER, G. M. (1975). Covalently linked message and anti-message (genomic) RNA from a defective vesicular stomatitis virus particle. J. Mol. Biol. 97, 289-308. LEAMNSON, R. N., and REICHMANN, M. E. (1974). The RNA of defective vesicular stomatitis virus particles in relation to viral cistrons. J. Mol. Biol. CARTWRIGHT,

85,551~568.

J. A., and REICHMANN, M. E. (1975). RNA synthesis by temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype. Virology 63, 492-504. MACPHERSON, I. A., and STOKER, M. G. P. (1962). Polyoma transformation of hamster cell clones An investigation of genetic factors affecting cell competence. Virology 16, 147-151. NAKAI, T., and HOWATSON, A. F. (1968). The fine structure of vesicular stomatitis virus. Virology

LESNAW,

35, 268-281. PERRAULT, J.

(1976). Cross-linked double stranded RNA from a defective vesicular stomatitis virus particle. Virology 70, 360-371. PERRAULT, J., and HOLLAND, J. J. (1972). Variability of vesicular stomatitis virus autointerference with different host cells and virus serotypes. Virology 50, 148-158. PETRIC, M., and PREVEC,

titis virus-A lar structures,

L. (1970). Vesicular stomanew interfering particle, intracelluand virus-specific RNA. Virology

500

SCHNITZLEIN

AND REICHMANN

41, 615-630. PREVEC, L. (1974). Physiological properties of vesicular stomatitis virus and some related rhabdoviruses. In “Viruses, Evolution and Cancer” (E. Kurstak and K. Maramorosch, eds.), pp. 677-697. Academic Press, New York. PREVEC, L., and KANG, C. Y. (1970). Homotypic and heterotypic interference by defective particles of vesicular stomatitis virus. Nature (London) 228, 25-27. PRINCLE, C. R., DUNCAN, I. B., and STEVENSON, M. (1971). Isolation and characterization of temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype. J. Viral. 8, 836-841. REICHMANN, M. E., PRINGLE, C. R., and FOLLETT, E. A. C. (1971). Defective particles in BHK cells infected with temperature-sensitive mutants of vesicular stomatitis virus. J. Viral. 8, 154-160. REPIIC, P., and BISHOP, D. H. L. (1973). Determination of the molecular weight of animal RNA viral genomes by nuclease digestions. J. Virol. 12, 969983. REPIK, P., FLAMAND, A., CLARK, H. F., OBIJESKI, J. F., ROY, P., and BISHOP, D. H. L. (1974). The detection of homologous RNA sequences among

six rhabdovirus genomes. J. Viral. 13,250-252. J. K., and KNIPE, D. (1975). Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species. J. Viral. 15, 994-1003. SCHAFFER, F. L., and SOERGEL, M. E. (1972). Molecular weight estimates of vesicular stomatitis virus ribonucleic acids from virions, defective particles, and infected cells. Arch. Gesamte Virusforsch. ‘39, 203-222. SCHNITZLEIN, W. M., and REICHMANN, M. E. (1976). The size and the cistronic origin of defective vesicular stomatitis virus particle RNAs in relation to homotypic and heterotypic interference. J. Mol. Biol. 101, 307-325. STAMMINGER, G., and LAZZARINI, R. A. (1974). Analysis of the RNA of defective VSV particles. Cell 3, 85-93. STAMPFER, M., BALTIMORE, D., and HUANG, A. S. (1971). Absence of interference during high multiplicity infection by clonally purified vesicular stomatitis virus. J. Viral. 7, 409-411. UNGER, J. T., and REICHMANN, M. E. (1973). RNA synthesis in temperature-sensitive mutants of vesicular stomatitis virus. J. Viral. 12, 570-578. ROSE,